Cyclin-dependent kinase 4 (Cdk4) is an important regulator of GI/S cell cycle progression of mammalian cells in culture. Germline mutations in the 24th codon of this gene (R to C) result in a predisposition of the individuals to the development of melanoma. To understand the role of this enzyme in vivo, we have generated strains of mice that either lack Cdk4 expression (Cdk4 net/net) or express an activated form of this enzyme (Cdk4 R24CIR24C), which cannot interact with the CDK Inhibitor pl6INK4A. Homozygous Cdk4 null mutant mice (Cdk4 net/net) are viable but express defects in growth, spermatogenesis and oogenesis. In addition, these mice were found to be diabetic exhibiting defective pancreatic g-cell development. On the other hand, Cdk4(R24C/R24C) mouse embryo fibroblasts (MEFs) display increased Cdk4-kinase activity resulting in hyperphosphorylation of all three members of the Rb-family, pRb, P107 and p130. These MEFs display decreased doubling times and escape from replicative senescence which appears to coincide with a loss of p21Wafl/Cipl expression. In addition, they also exhibit a high degree of susceptibility to oncogene-induced transformation, suggesting that the Cdk4R24C mutation can serve as a primary event in the progression towards a fully transformed phenotype. In agreement with this, Cdk4(R24C/R24C) mice develop tumors of varying etiology within 8-10 months of their life span. To gain an insight into the role of Cdk4 in cell cycle regulation, neoplasia, and senescence, we propose: (1) To carry out a detailed characterization of MEFs derived from Cdk4 (R24C/R24C) mice to understand the molecular basis for their ability to escape senescence (2) To carry out a detailed expression analysis of the Kip family members as a function of cell passage and determine the effect of their loss on CDK2 enzyme activity in Cdk4 (R24C/R24C) MEFs that become immortalized in vitro and in naturally occurring tumors that develop in Cdk4 (R24C/R24C) animals vivo; (3) to examine the molecular basis for the loss of p21 expression in Cdk4(R24C/R24C) MEFs and examine the genetic consequence of loss of p21 expression in cells that harbor Cdk4 mutation on cellular senescence in vitro and tumor development in vivo; and (4) to test whether Cdk4 is required for breast tumor development induced by the oncogenic pathways mediated by ras, neu, myc and Wnt-1 expressed under the control of MMTV-LTR.